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. 2016 May 19;11(5):e0155711. doi: 10.1371/journal.pone.0155711

Fig 3. The effect of vorinostat on the sensitivity of cancer cells to ABT-888 is mediated by increased phosphorylation of eIF2α.

Fig 3

a. Vorinostat increased eIF2α phosphorylation in ABT-888 treated MDA-MB-231 cells but not in ABT-888 treated fibroblasts and normal breast cells: ABT-888 (10 μM), vorinostat (0.5 μM) and their combination were added to the cells 48 hours post-plating. The cells were harvested 48 hours later and processed for western blot analysis of changes in the ratio p-eIF2α/eIF2α. Numbers at the bottom of the autoradiograms indicate changes relative to controls of p-eIF2α/ eIF2α and of loaded proteins (Ponceau). The experiment was reproduced once with similar results. b. Salubrinal increases CD of MDA-MB-231 cells in response to ABT-888: Cells were treated with salubrinal, ABT-888 or both. Values are means CD (%) of triplicates ± SEM. The experiment was reproduced once with similar results. Differences between CD (%) of combined treatment and each of the experimental treatments or controls were significant. **p<0.01, * p<0.05. CI were < 0.9. c. A phosphomimetic eIF2α variant increases clonogenic death in response to ABT-888: The PARPi (10 μM) was added to the cells 18 hours following transfection with 1.5μg/2ml eIF2α S51A or eIF2α S51D. Colonies were processed for analysis 10 days post-treatment initiation. Values are means CD (%) of triplicate samples ± SEM. The experiment was reproduced twice with similar results. Differences between each of the controls and their ABT-888 treated counterparts as well as between the ABT-888 treated S51A and S51D variants were significant. SA—non-phosphorylatable S51A eIF2α, SD—phosphomimetic S51D eIF2α. d. Salubrinal increases eIF2α phosphorylation and 53BP1 expression in ABT-888 treated cells without affecting the level of BRCA1 or RAD51: ABT-888 (10 μM) and salubrinal (4.5 μM) were added to the cells 48 hours post-plating. The cells were harvested 48 hours later and processed for western blot analysis of changes in BRCA1, RAD51, 53BP1 and the ratio p-eIF2α/eIF2α. Numbers at the bottom of the autoradiograms are changes relative to controls of the level of BRCA1, RAD51, 53P1, the ratio p-eIF2α/eIF2α and the amounts of loaded proteins (Ponceau). The experiment was reproduced once with similar results. e. The level of phosphorylated eIF2α is higher and the level of total eIF2α is lower in normal fibroblasts and breast cells than in cancer cells: Cells were harvested for western blot analysis four days post-plating. The experiment was reproduced once with similar results. Numbers at the bottom of the autoradiograms indicate changes relative to control of peIF2α/eIF2α and the amount of loaded proteins (Ponceau). The experiment was reproduced once with similar results. Unabridged images of the MDA autoradiogram in panel (a) of RAD51 and 53BP1 in panel (d) and of the autoradiograms in panel (e) are presented in S3 Fig.